【作者】 肖剑锋；

【导师】 王勇； 沈建新；

【作者基本信息】 汕头大学 ， 生理学， 2008， 硕士

【摘要】 背景:Ca2+是细胞内最严格的调节离子,参与了各种Ca2+结合蛋白的活动,例如激酶、磷酸酶和蛋白激酶的信号级联激活。钙信号诱导各种生理反应,参与包括肌肉收缩、囊泡分泌、细胞分化、增殖和细胞死亡。不同细胞对钙信号的反应不同,在同一细胞中,不同形式的钙信号可产生不同的反应。例如在心肌细胞,动作电位导致的Ca2+内流触发细胞收缩;而在病理生理条件下(如心肌肥大),钙信号可通过基因表达激活转录因子,诱导心肌表型重构。此外,钙信号延长可以导致细胞死亡。因此,明确不同的钙信号系统是如何控制不同的细胞功能对于更好的理解机体组织中钙信号的转导有重要意义。Ca2+作为细胞信号存在于细胞外液和胞内的储存库,前者例如许多胞浆膜通道受到膜去极化、胞外和胞内配体、机械牵拉刺激引起通道开放,后者例如ryanodine受体和IP3受体对从内质网或者肌浆网释放的Ca2+产生反应。对心肌细胞来说,离子钙将细胞的兴奋(即动作电位)与细胞的收缩联系起来,此即为兴奋-收缩耦联(excitation-contraction coupling, ECC)。在ECC过程中,心肌细胞的兴奋引起胞膜上的电压依赖性L-型钙通道开放,胞外Ca2+内流,以钙诱导钙释放(Ca2+-induced Ca2+ release, CICR)的方式引起心肌细胞肌浆网(sarcoplasmic reticulum, SR)在短时间内通过其膜上的钙释放通道(即ryanodine受体)向胞浆释放大量的钙,造成胞浆内钙离子浓度瞬时升高,此即为钙瞬变(Ca2+ transients)。钙瞬变通过肌钙蛋白(troponin)引起细胞收缩[1]。钙瞬变是心肌细胞中最明显、最强烈和最典型的胞内钙信号变化。通过对钙瞬变的观测,可了解心肌细胞的功能活动特点和状态。因此,我们以共聚焦显微镜作为记录方式,通过给予不同的药物灌流作用于心室肌细胞,以局部场电刺激的方法诱发全细胞的钙释放(钙瞬变),观察不同的药物对心室肌细胞胞内钙信号的影响,并探讨其发生机制为临床心血管疾病的防治提供一定的理论依据。目的:(1)观测不同浓度的高钾灌流液对成年SD大鼠心室肌细胞胞内钙信号影响,分析图像数据中所包含的信息,探讨不同浓度的高钾灌流液影响心室肌细胞胞内钙信号可能的机制;(2)观测不同浓度的低钾灌流液对成年SD大鼠心室肌细胞胞内钙信号影响,分析图像数据中所包含的信息,探讨不同浓度的低钾灌流液影响心室肌细胞胞内钙信号可能的机制;(3)观测不同浓度的钙通道阻滞剂-硝苯地平灌流液对成年SD大鼠心室肌细胞胞内钙信号影响,分析图像数据中所包含的信息,探讨不同浓度的硝苯地平灌流液影响心室肌细胞胞内钙信号可能的机制。材料与方法:清洁级成年SD大鼠,雌雄不拘,体重200~350g。采用改进的Langendorff装置,行主动脉插管逆向灌流,以0.6mg/ml胶原酶Ⅱ+ 0.06mg/ml蛋白酶+1mg/ml牛血清白蛋白混合液消化心脏,经三次不同浓度含钙台氏液复钙后得到钙稳态心肌细胞。室温放置1~2小时后将分离好的心肌单细胞先与钙荧光指示剂fluo-4-AM(终浓度为15μmol/L)(Molecular probes,Inc)共同孵育15min,弃上清去除细胞外钙荧光染料后加入台氏液。适合进行实验的细胞必需符合以下标准:①横纹肌清晰;②呈杆状;③表面干净平整;④观察1min无自发收缩活动。孵育好染料的细胞置于Zeiss LSM-510 META型共聚焦显微镜的载物台上进行观测。共聚焦显微镜钙成像主要采用快速二维扫描(fast 2-dimension scanning)和线扫描(line scanning)二种方式,通过触发连接可使SEN-7203型电子刺激器与LSM-510型共聚焦显微镜同步工作得到心肌细胞的诱发钙瞬变图像。实验获得的激光共聚焦显微镜图像采用自编程序处理,编程语言为IDL(interactive data language, research systems, Inc.)钙瞬变的幅度以标准荧光强度的净变化△R=△F/F0表示,其中F0为静息状态下fluo-4的背景荧光强度。结果:1.高钾对心室肌细胞胞内钙信号的影响:(1)正常细胞外液时(胞外钾离子浓度为5.4mmol/L)心肌细胞在线扫描(line scanning)模式下钙瞬变峰值为7.56±0.56(单位为△F/F0),给予7.5mmol/LKCl灌流后,心肌细胞钙瞬变峰值为7.90±0.63(单位为△F/F0),标准化后比值为1.07±0.03大于1,表明其胞内钙瞬变峰值比正常液强(P<0.05);给予10、20、50mmol/L KCl的细胞外液灌流,心肌细胞钙瞬变峰值均较正常液降低,并且随着细胞外液KCl浓度增高而逐渐降低(P<0.05)。(2)给予不同浓度的高钾(7.5、10、20、50mmol/L KCl)灌流心室肌细胞,静息状态下心室肌细胞胞内钙信号随着钾离子浓度的升高而增高(P<0.05),在50mmol/L KCl灌流时,心室肌细胞胞内钙信号强度约为正常液灌流时的3倍,心室肌细胞胞内钙稳态处于失衡状态。2.低钾对心室肌细胞胞内钙信号的影响:(1)给予2mmol/L KCl的细胞外液灌流心肌细胞(共观察6个细胞),5个心肌细胞可正常诱发钙瞬变,所诱发的钙瞬变峰值(反映钙释放的强度)为6.85±0.70与对照组6.54±0.21相比变化不明显(P>0.05);半高宽(反映钙释放持续时间)为203.31±17.12 ms,与对照组176.46±17.08 ms相比,明显延长(P<0.05)。洗去2mmol/L KCl之后诱发钙瞬变的峰值为6.25±0.28,与对照组比略有降低,但P>0.05;半高宽222.49±17.00 ms,与对照组比,明显延长(P<0.05),这说明2mmol/L低钾液可延长诱发钙瞬变的持续时间,且此效应在短时间内不易完全恢复。另有1个心肌细胞先有正常诱发钙瞬变,后对场刺激不反应,然后出现自发钙瞬变。(2)给予1mmol/L KCl的细胞外液灌流心室肌细胞(共观察9个细胞),5个心肌细胞可正常诱发钙瞬变,所诱发的钙瞬变峰值(反应钙释放的强度)虽有增大趋势,但P>0.05;洗去1mmol/L低钾液之后半高宽为209.0±22.1 ms与对照组178.4±18.8 ms比较明显延长(P<0.05)。1个心肌细胞先有正常诱发钙瞬变(时程延长),后对场刺激无反应,后出现自发现象;1个心肌细胞刚开始对场刺激无反应,后有正常诱发钙瞬变(时程延长),后又无反应;2个心肌细胞对场刺激不反应,只有自发钙瞬变。(3)给予无钾的细胞外液灌流心室肌细胞(共观察8个细胞),7个细胞有自发现象,洗去无钾液之后诱发钙瞬变恢复,但效应时程211.0±9.7 ms较对照组155.4±3.8 ms均延长(P<0.05):其中的3个细胞先有正常诱发钙瞬变(时程延长),后出现自发钙瞬变或钙波现象;3个细胞刚开始无反应(对场刺激不反应),后产生自发钙瞬变或钙波;1个细胞自发钙瞬变或钙波(对场刺激不反应);1个细胞无自发反应,诱发钙瞬变在灌流后迅速明显减弱,后诱发钙瞬变强度恢复到接近正常,但时程明显延长,细胞收缩基本消失。3.不同浓度硝苯地平对心室肌细胞胞内钙信号的影响:所有浓度水平(2、5、10、50μmol/L)的硝苯地平都可使大鼠心室肌细胞胞内钙瞬变峰值降低(P<0.05),在所检测的各浓度水平中,5μmol/L硝苯地平标准化值最低,降低胞内钙瞬变峰值的效果比2μmol/L硝苯地平明显(P<0.05)。硝苯地平对静息状态下大鼠心室肌细胞胞内钙信号的影响则不明显。结论:1.胞外高钾液灌流可引起心室肌细胞胞内钙稳态失衡,随着胞外钾离子浓度的升高,心室肌细胞胞内钙瞬变峰值先升高后降低;重度钾离子浓度(10、20、50mmol/L)升高所引起的诱发钙瞬变峰值降低与相应状态下胞内静息钙水平的升高有密切联系。2.尽管心室肌细胞不同个体对胞外低钾的敏感性不同,但总的趋势是:低钾可以引起心室肌细胞胞内钙稳态失衡。轻度低钾液灌流引起诱发钙瞬变的渐变(峰值不变而钙释放时间延长),无钾液灌流可引起胞内钙信号的突变:出现自发钙瞬变或钙波。3.硝苯地平可降低心室肌细胞胞内钙瞬变强度,但是不影响心室肌细胞胞内的静息钙水平。更多还原

【Abstract】 Background:Ca2+ is one of the most strictly regulated ions in the cell, because it is involved in many important signal cascades by activating various Ca2+-binding proteins, such as kinases, phosphatases, and proteases. Ca2+ signals induce various physiological responses such as muscle contraction, vesicle secretion, cell differentiation, proliferation, and cell death. Whereas some of the responses to the Ca2+ signal are cell-type specific, multiple Ca2+ signals are used for different responses in the same cell. In cardiac myocytes, for example, Ca2+ triggers muscle contraction in response to every action potential, whereas in pathophysiological circumstances (e.g. cardiac hypertrophy), the Ca2+ signal induces phenotypic remodeling of the myocytes via gene expression by activating transcription factors. Moreover, the prolonged Ca2+ signal leads to cell death. It is thus important to clarify how different Ca2+ signal systems can be set up to control different cellular functions for the better understanding of the Ca2+ signal transduction in native tissues. Ca2+ used for cell signaling is derived either from the extracellular fluid or internal stores. In the former case, many kinds of plasma membrane depolarization, extracellular and intracellular ligands, and mechanical stretch. In the latter case, ryanodine receptor (RyR) and inositol-1,4,5-triphosphate (InsP3) receptor are responsible for the release of Ca2+ from the internal Ca2+ store such as the endoplasmic reticulum (ER) or its muscle equivalent, the sarcoplasmic reticulum (SR). The excitation of cells (action potential) was connected with the contraction of cells through Ca2+ which is called excitation-contraction coupling, ECC. Ca2+ influx through the L-type Ca2+ channels which were caused open by the excitation of myocyte result the sarcoplasmic reticulum Ca2+ channels open, a large of Ca2+ release in the form of Ca2+-induced Ca2+ release. The concentration of Ca2+ intracytoplasm burst at a short time which is called calcium transients. Calcium transients cause the cell contracting through the troponin, which is the most obvious, stronger and typical calcium signal change intracellular. We can understand the function, activity, characteristic and state of the myocytes by observing the calcium transient. So we record the calcium transients of rat ventricular myocytes which were induced by local field stimulation in different drug circumstance with the help of confocal system Zeiss LSM510 META. Discussing the mechanism and hope it can provide some theory for the cardiovascular disease therapy.Objective:(1) Observing the changes of calcium signals about the ventricular myocytes of adult SD rats in different hyperkalemia, analysising the information in the images and discussing the possible mechanism which affect the calcium signals of the ventricular myocytes about adult SD rats in different hyperkalemia.(2) Observing the changes of calcium signals about the ventricular myocytes of adult SD rats in different hypokalemia, analysising the information in the images and discussing the possible mechanism which affect the calcium signals of the ventricular myocytes about adult SD rats in different hypokalemia.(3) Observing the changes of calcium signals about the ventricular myocytes of adult SD rats in different concentration of nifedipine, analysising information the in the images and discussing the possible mechanism which affect the calcium signals of the ventricular myocytes about adult SD rats in different concentration of nifedipine.Materials and methods:Adult Sprague-Dawley rats of either sex (weight, 200-350g,) were anesthetized with sodium pentobarbital (50 mg/kg IP). Once anesthetized, the heart was rapidly excised and mounted on a Langendorff perfusion apparatus. Ventricular myocytes were dispersed by enzymatic (0.6mg/ml collagenase; typeⅡ+0.06mg/ml protease+1mg/ml BSA) digestion. Enzymatically isolated ventricular myocytes were loaded with Ca2+ indicator Fluo-4-AM (15μmol/L) for 15 min, followed by a 10 min rest allowing for deesterfication of the indicator. The criteria for cell selection included rod shape, clear striation and clean cell surface, and lack of spontaneous contractions during a 1-min observation period. Ca2+ images were acquired by using a Zeiss LSM510 confocal microscopy. Computer programs for the calcium transients were coded in Interactive Data Language (IDL, Research Systems, Boulder, CO). Calcium transients were measured as△R =△F/F0 , where F refers to the present Fluo signal intensity, F0 the background Fluo signal intensity, and△F/F0 the alteration of F/F0. Results:1. The changes of calcium signals on the circumstance of hyperkalemia: (1) the peak of calcium transients is 7.56±0.56 (△F/F0) at the condition of 5.4mmol/L KCl, line scanning. The peak of calcium transients is 7.90±0.63 (△F/F0) after perfusing with 7.5mmol/LKCl. The value of normalization is 1.07±0.03>1, the peak of calcium transients become higher than the control; The peak of calcium transients become lower step by step after perfusing with 10、20、50 mmol/L KCl (P<0.05). (2) The background calcium signals become higher step by step after perfusing with 10、20、50mmol/L KCl (P<0.05).The background calcium signals is triplicity than the control, the calcium homeostasis was broken intracellular ventricular myocytes.2. The changes of calcium signals on the circumstance of hypokalemia:(1) the peak of calcium transients (6.85±0.70) which induced from 5 ventricular myocytes (observing 6 ventricular myocytes) after perfusing with 2mmol/L KCl is not significant, compared with the control (6.54±0.21) (P>0.05); FWHM (meaning the time course of calcium release) is 203.31±17.12 ms on the circumstance of 2 mmol/L KCl, which is significant compared with the control 176.46±17.08 ms (P<0.05). The peak of calcium transients reduce slightly comparing with the control (P>0.05), FWHM 222.49±17.00 ms prolonged obviously than the control (P<0.05) after perfusing with the normal extracellular fluid. It indicated that 2mmol/L KCl can induce the prolongation of the time course about calcium transients, and the effects can not recover at a short time. We observed a ventricular myocyte can induced calcium transients at first, then have no reaction to the local stimulation and appear calcium transients in the form of spontaneous.(2) We observed 9 ventricular myocytes at 1 mmol/L KCl, 5 cells can induce calcium transients, the peak increase but P>0.05. FWHM 209.0±22.1 ms after washing out 1 mmol/L KCl with normal extracellular fluid prolonged significantly compared with the control 178.4±18.8 ms (P<0.05). 1 cell can induce calcium transients (the time course prolonged), then has no reaction to the local stimulation, then appear calcium transients in the form of spontaneous: 1 cell has no reaction to the local stimulation at first, then can induce calium transients, then has no reaction to the local stimulation; 2 cells have no reaction to the local stimulation, appearing calcium transients in the form of spontaneous.(3) We observed 8 ventricular myocytes at the circumstance of 0 mmol/L KCl. 7 cells appear calcium transients in the form of spontaneous, then the induce calcium transients recover after perfusing with normal extracellular fluid but the time course 211.0±9.7 ms prolong than the control 155.4±3.8 ms; 3 cells can induce the calcium transients to the local stimulation at first (the time course prolong), then appear calcium transients or calcium wave in the form of spontaneous; 3 cells have no reaction to the local stimulaton then appear calcium transients or calcium wave in the form of spontaneous; 1 cell appear calcium transients or calcium wave in the form of spontaneous;1 cell calcium transients reduce quickly, then recover, but the time course prolong, the cell do not contract.3. The changes of calcium signals on the different concentration of nifedipine: The peak of calcium transients reduced at the different concentration of nifedipine. The value of normalization on the 5μmol/L nifedipine is the lowest, the effect is better than the 2μmol/L nifedipine (P<0.05). The background calcium signals haves no obvious change on the different concentrations of nifedipine.Conclusions:1. Hyperkalemia can break the homeostasis of intracellular calcium about ventricular myocytes, the peak of calcium transients increase at first then reduce with the [K+]o increasing.The depression about the peak of calcium transients has correlated to the increase about the background calcium signals closely when [K+]o increasing (10、20、50mmol/L).2. The different cells have different reaction to the hypokalemia, but the tendency is that hypokalemia can disturb the homeostasis of intracellular calcium about ventricular myocytes. The reduce slightly of [K+]o cause the change of calcium transients step by step (the peak do not change , the time course of calcium release prolong), the 0mmol/L KCl cause the calcium signals of ventricular myocytes jumping change, we can observe the calcium transients or calcium waves in the form of spontaneous.3. Nifedipine can reduce the magnitude of calcium transients, but have no effects on the background calium signals about ventricular myocytes.更多还原